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Methods for Single-Molecule Sensing and Detection Using Bacteriophage Phi29 DNA Packaging Motor

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Molecular Motors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1805))

Abstract

Bacteriophage phi29 DNA packaging motor consists of a dodecameric portal channel protein complex termed connector that allows transportation of genomic dsDNA and a hexameric packaging RNA (pRNA) ring to gear the motor. The elegant design of the portal protein has facilitated its applications for real-time single-molecule detection of biopolymers and chemicals with high sensitivity and selectivity. The robust self-assembly property of the pRNA has enabled biophysical studies of the motor complex to determine the stoichiometry and structure/folding of the pRNA at single-molecule level. This chapter focuses on biophysical and analytical methods for studying the phi29 motor components at the single-molecule level, such as single channel conductance assays of membrane-embedded connectors; single molecule photobleaching (SMPB) assay for determining the stoichiometry of phi29 motor components; fluorescence resonance energy transfer (FRET) assay for determining the structure and folding of pRNA; atomic force microscopy (AFM) for imaging pRNA nanoparticles of various size, shape, and stoichiometry; and bright-field microscopy with magnetomechanical system for direct visualization of viral DNA packaging process. The phi29 system with explicit engineering capability has incredible potentials for diverse applications in nanotechnology and nanomedicine including, but not limited to, DNA sequencing, drug delivery to diseased cells, environmental surveillance, and early disease diagnosis.

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Acknowledgments

The research was supported by NIH grants U01-CA151648 and R01-EB012135. Funding to P.G.’s Endowed Chair in Nanobiotechnology position is by the William Fairish Endowment Fund. P.G. is a cofounder of Biomotor and Nucleic Acid Nanotechnology Development Corp., Ltd.

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Authors and Affiliations

  1. Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA

    Farzin Haque, Hui Zhang, Shaoying Wang & Peixuan Guo

  2. Department of Physiology and Cell Biology, Dorothy M Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA

    Farzin Haque, Hui Zhang, Shaoying Wang & Peixuan Guo

  3. Nanobiotechnology Center, University of Kentucky, Lexington, KY, USA

    Farzin Haque, Hui Zhang, Shaoying Wang & Peixuan Guo

  4. Markey Cancer Center, University of Kentucky, Lexington, KY, USA

    Farzin Haque, Hui Zhang, Shaoying Wang & Peixuan Guo

  5. Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, USA

    Farzin Haque, Hui Zhang, Shaoying Wang & Peixuan Guo

  6. Birck Nanotechnology Center, Purdue University, West Lafayette, IN, USA

    Chun-Li Chang & Cagri Savran

  7. School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA

    Chun-Li Chang & Cagri Savran

  8. Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA

    Chun-Li Chang & Cagri Savran

  9. Sylvan G. Frank Endowed Chair in Pharmaceutics and Drug Delivery, The Ohio State University, Columbus, OH, USA

    Peixuan Guo

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  1. Farzin Haque
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  2. Hui Zhang
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  3. Shaoying Wang
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  4. Chun-Li Chang
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  5. Cagri Savran
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  6. Peixuan Guo
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Correspondence to Peixuan Guo .

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Editors and Affiliations

  1. National Museum of Natural History, CNRS UMR7196 / INSERM U1154, Paris, France

    Christophe Lavelle

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Haque, F., Zhang, H., Wang, S., Chang, CL., Savran, C., Guo, P. (2018). Methods for Single-Molecule Sensing and Detection Using Bacteriophage Phi29 DNA Packaging Motor. In: Lavelle, C. (eds) Molecular Motors. Methods in Molecular Biology, vol 1805. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8556-2_21

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  • DOI: https://doi.org/10.1007/978-1-4939-8556-2_21

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